The natural pigment carminic acid is one of the most frequently used colorants of food, medicine, cosmetics and textiles.
Carminic acid is a colorant, which can be extracted from the female insect bodies of Dactylopius coccus costa (alternative name Coccus cacti L.). The insects live on Nopalea coccinellifera, Opuntia fidus indica and other plants of the family Cactaceae cultivated for instance in the desert areas of Mexico, Central and South America and Canary Islands. Depending on the pH the colorant may be a color in a spectrum from orange over red to purple and is generally known as cochineal or cochineal color. Carmine colorant is widely used in foods and beverages.
As known in the art Porphyrophora polonica is also producing carminic acid and was cultured for production of carminic acid in e.g. Poland.
In relation to current industrial relevant production, carminic acid is harvested by extraction from the insect's dried bodies with water or alcohol.
In order to try to resolve the problem of undesirable variations and price fluctuations—U.S. Pat. No. 5,424,421 (European Colour, published 1995) describes chemical synthesis of carminic acid by a route of synthesis involving different intermediates.
As discussed in e.g. WO2006/056585A1 (Chr. Hansen A/S), during the aqueous based extraction of carminic acid from the insect, an amount of insect protein is also released from the insect and will be contained in the color extract and it has been reported that the cochineal insect proteins could create some allergy related problems. In WO2006/056585A1 a special process to reduce the amount of insect protein from the insect extract solution is described. However, the final produced color composition/product of WO2006/056585A1 will still comprise some amounts Dactylopius coccus costa insect proteins.
The structure of carminic acid is shown in FIG. 1 herein. As can be seen from the Figure, it is a so-called C-glucoside (i.e. wherein the glucose is joined/conjugated to the aglucon by a carbon-carbon linkage).
As shown in FIG. 1 herein, hydrolysis of the C-glucoside carminic acid can give glucose and the aglucon kermesic acid (KA).
The in vivo biosynthetic pathway of carminic acid in the insect (Dactylopius coccus) is currently not described in details. Accordingly, based on the prior art the skilled person does not know which compound is the aglucon during the in vivo biosynthetic production of carminic acid in Dactylopius coccus. 
Analysis of Dactylopius coccus has shown that a broad range of compounds related to carminic acid are present in extracts from Dactylopius coccus and numerous of these compounds could in principle be glucosylated during the in vivo biosynthetic production of carminic acid.
For instance, in the article of Stathopoulou et al. (Analytica Chimica Acta 804 (2013) 264-272) six new anthraquinones were described in an extract from D. coccus and any of these six new anthraquinones (see e.g. FIG. 1 of the article) could in principle be the molecule which is glucosylated during the in vivo biosynthetic production of carminic acid in Dactylopius coccus. Further, as known in the art the primary glucosylated compound formed during the in vivo biosynthetic production of the glucoside end product may be an unstable intermediate compound that will not be identified in an isolated extract from Dactylopius coccus as e.g. analyzed in the above discussed article of Stathopoulou et al.
As understood by the skilled person in the present context, based on the prior art, it could be speculated that a relevant primary glucosylated compound during the in vivo biosynthetic production of carminic acid in Dactylopius coccus could e.g. be an unstable intermediate polyketide compound with around the same number of carbon atoms as e.g. flavokermesic acid.
A herein relevant DNA or amino acid sequence of a glycosyltransferase involved in the in vivo insect (Dactylopius coccus) biosynthetic pathway of carminic acid is not explicitly described in the prior art.
As known in the art, for insects that accumulate low molecular weight chemicals the relevant biosynthetic pathway genes are sometimes not present in the insect genome. For instance, some insects take up glycosides from the plants they feed on—see e.g. the article of Zagrobelny et al (Cyanogenic glucosides and plant-insect interactions; Phytochemistry. 2004 February; 65(3):293-306) or the article of Geuder et al (Journal of Chemical Ecology, Vol. 23, No. 5, 1997).
Dactylopius coccus insects feed on cactus plants and it could be that Dactylopius coccus insects (like other insects) take up relevant glycosides from the cacti they feed on. As known in the art, for insects that accumulate low molecular weight glycosides, the relevant biosynthetic pathway genes are sometimes found in the microorganisms living in the insects, see e.g. the article of Genta et al. (Potential role for gut microbiota in cell wall digestion and glucoside detoxification in Tenebrio molitor larvae), Journal of Insect Physiology 52 (2006) 593-601.
Accordingly, based on the prior art the skilled person could not know if the genome of Dactylopius coccus actually would comprise a gene encoding a glycosyltransferase involved in the in vivo biosynthetic pathway leading to carminic acid.
Polyketides are synthesized by a group of enzymes which commonly is referred to as polyketide synthases (PKS). All PKSs share the ability to catalyze Claisen condensation based fusion of acyl groups by the formation of carbon-carbon bonds with the release of carbon dioxide. This reaction is catalyzed by a beta-ketosynthase domain (KS). In addition to this domain/active site, synthesis can also depend on, but not exclusively, the action of Acyl-Carrier-Protein (ACP), Acyl-transferase (AT), Starter-Acyl-Transferase (SAT), Product Template (PT), ThioEsterase (TE), Chain Length Factor (CLF, also known as KSβ), Claisen CYClase (CYC), Ketoreductase (KR), dehydratase (DH), enoyl reductase (ER) and C-methyl transferase (Cmet). The substrates for polyketide synthesis are typically classified into starter and extender units, where the starter unit, including but not limited to acetyl-CoA is the first added unit of the growing polyketide chain; and extender units, e.g. but not exclusively malonyl-CoAs, are all subsequently added carbon-carbon units. At the primary sequence level (amino acid sequence), secondary structure level (local fold), tertiary structure level (all over fold) and quaternary structure level (protein-protein interactions) the PKSs display a very large diversity, and are hence subdivided into different types.
Type I PKS systems are typically found in filamentous fungi and bacteria, where they are responsible for both the formation of aromatic, polyaromatic and reduced polyketides. Members of the type I PKS possess several active sites on the same polypeptide chain and the individual enzyme is able to catalyze the repeated condensation of two-carbon units. The minimal set of domains in type I PKS includes KS, AT and ACP. The type I PKSs is further subdivided into modular PKSs and iterative PKSs, where iterative PKSs only possess a single copy of each active site type and reuse these repeatedly until the growing polyketide chain has reached its predetermined length. Type I iterative PKS that forms aromatic and polyaromatic compounds typically rely on the PT and CYC domain to direct folding of the formed non-reduced polyketide chain. Modular PKSs contain several copies of the same active sites, these are organized into repeated sequences of active sites which are called modules, each module is responsible for adding and modifying a single ketide unit. Each active site in the individual modules is only used once during synthesis of a single polyketide. Type I iterative PKS are typically found in fungi, while type I modular PKSs are typically found in bacteria.
Type II PKS systems are responsible for formation of aromatic and polyaromatic compounds in bacteria.
Type II PKSs are protein complexes where individual enzymes interact to form the functional PKS enzyme. The individual enzymes include activities for KS, CLF and ACP. This type of PKS is characterized by being composed of multiple different enzymes that form a protein complex, which collectively is referred to as an active PKS. The type II PKSs form non-reduced polyketides that spontaneously folds into complex aromatic/cyclic compounds. However, in the bacterial systems folding of polyketide backbones is most often assisted/directed by different classes of enzymes, that act in trans (independent of the PKS enzyme) to promote a non-spontaneous fold. The involved enzyme classes are referred to as aromatases and cyclases. The biosynthesis of a single polyaromatic compound in these systems typically involves the successive action of multiple different aromatases/cyclases. The aromatases and cyclases can be divided into two groups based on which types of substrates they act on: where the first group only acts on linear polyketide chains and catalyzes formation of the first aromatic/cyclic group, the second group of enzymes only accepts substrates that include aromatic or cyclic groups (=products from the first group of aromatases/cyclases). It has proven impossible to functionally express type II PKS systems in a suitable production host (E. coli, Bacillus, yeast), likely due to the fact that these are multienzyme complexes which require a balanced expression level, and which may rely on unknown factors.
Type III PKSs generally only consist of a KS domain, which in the literature may e.g. be referred to as a KASIII or a Chalcone synthase domain that acts independently of the ACP domain. Type III PKS from bacteria, plant and fungi have been described.
Type III PKSs have long been known in plants, where they are responsible for formation of compounds such as flavonoids (pigments/anti-oxidants) and stilbenes, which are found in many different plant species. The products of type III PKSs often spontaneously folds into complex aromatic/cyclic compounds.
The article of Yu et al. (2012) provides a review of Type III Polyketide synthases in natural product biosynthesis. The Yu et al. (2012) article reads: “Type III PKSs are selfcontained enzymes that form homodimers. Their single active site in each monomer catalyzes the priming, extension, and cyclization reactions iteratively to form polyketide products. Despite their structural simplicity, type III PKSs produce a wide array of compounds such as chalcones, pyrones, acridones, phloroglucinols, stilbenes, and resorcinolic lipids. In recent years, type III PKSs have drawn more attention due to their diverse products, wide distribution, relatively simple structures, and easy genetic manipulability. In this article, we will systematically discuss type III PKSs from plants, bacteria, and fungi as well as the recent progress in the type III PKS research.”
In short, based on the prior art, the skilled person knows if a specific PKS of interest is a Type I, Type II or Type III PKS.
In addition to the protein structural and functional based classification of PKS systems, an alternative classification is based on the level of modifications found in the final polyketide product. Note that these modifications can either be introduced by the PKS itself or by post-acting enzymes. In this classification scheme the products are divided into two groups: (I) non-reduced and (II) reduced polyketides. The non-reduced type is characterized by the presence of ketone groups in the ketides (—CH2-CO—), originating from the starter or extender units, either as ketones or in the form of double bonds in aromatic groups. In reduced polyketides a single or all ketones have been reduced to alcohol (—CH2-CHOH—) groups by a KR domain/enzyme, or further to an alkene group (—C═C—) by a DH domain/enzyme, or even further to an alkane group (—CH2-CH2-) by an ER domain/enzyme. Based on these chemical features of the formed products the involved PKSs are categorized as either being a non-reducing PKS or a reducing PKS.
Folding of the formed polyketide chain into complex structures with cyclic motifs is typically a post-PKS enzyme guided and catalyzed process. The responsible enzymes belong to several different enzyme families, typically aromatases and/or cyclases. Fungal Type I iterative PKSs are special by posing a PT domain which is responsible for the formation of aromatic rings while CYC domains are responsible for product release coupled to formation of aromatic rings. The aromatases and cyclases acting on polyketides have been described from bacterial and plant systems. In addition, several examples exist where folding of the polyketide is a spontaneous process, e.g. flavonoids in plants.
PKSs have been isolated and functionally characterized from bacteria, fungi and plants. However, no PKS of animal origin has been described, and synthesis of polyketides in insects has in several instances been linked to the metabolic activity of endosymbiotic bacteria.
The article of Tang, Y. et al. (2004) describes that expression in the bacteria Streptomyces coelicolor CH999 strain, which contains chromosomal deletion affecting the entire Act gene cluster responsible for actinorhodin biosynthesis. The mini PKS (Act PKS=Act KS, Act CLF and Act ACP), belonging to the type II PKSs, yields flavokermesic acid (FK) (called TMAC in bacterial articles) when combined with heterologous expression of the ZhuI aromatase/cyclase and ZhuJ cyclase from the zhu gene cluster in Streptomyces sp. R1128. Accordingly, this article describes recombinant introduction of a Streptomyces PKS gene into a Streptomyces host cell, so the PKS is not of a different genus than the host cell.
In FIG. 2 herein is shown FIG. 5A of the Tang, Y. et al (2004) article. As can be seen in the figure and as further described in the article, the Act PKS (termed octaketide synthase (OKS)) creates a non-reduced octaketide and this octaketide is via the ZhuI aromatase/cyclase and ZhuJ cyclase converted into flavokermesic acid (FK) (called TMAC). The SEK4 and SEK4B compounds are also spontaneously produced (structures shown in FIG. 2 may herein be termed shunt products).
In the plant Aloe arborescens, identified PKSs have been shown to produce polyketides of various lengths including octaketides, see e.g. Mizuuchi et al (2009) where it in FIG. 1 is illustrated that the octaketide synthases (OKSs) termed PKS4 and PKS5 may, by using malonyl-CoA as extender units, create a non-reduced octaketide. The SEK4 and SEK4B shunt compounds are also spontaneously formed.
The plant Hypericum perforatum (St. John's wort) also comprises octaketide synthases, see e.g. Karppinen et al (2008), where it is described that the PKS termed HpPKS2 was expressed in E. coli, followed by purification and in vitro biochemical characterization of the enzyme. In FIG. 1 of the article is illustrated that the PKS termed HpPKS2 creates a non-reduced octaketide (using acetyl-CoA as starter unit and malonyl-CoA as extender units) and the shunt products SEK4 and SEK4B are spontaneously formed.
The article of Yu et al. (2012) provides a review of Type III Polyketide synthases in natural product biosynthesis; the article reads on page 293: “Various type III PKSs have been engineered into E. coli to generate novel polyketides. The production of plant-specific curcuminoids has been reconstituted in E. coli by co-expressing CUS with phenylalanine ammonia-lyase from Rhodotorula rubra and 4-coumarate:CoA ligase (4CL) from Lithospermum erythrorhizon”. As explained in the article, the PKS termed “CUS” synthesizes a diketide-CoA and therefore CUS is not an octaketide synthase.
The article Jadhav et al (2014) describes that a type III hexaketide PKS from Plumbago zeylanica (PzPKS) was cloned and expressed in tobacco plants to study whether the transgenic tobacco plants expressing PzPKS synthesize the pharmacologically important polyketide, plumbagin.
In none of the above mentioned PKS related articles are discussed production of carminic acid.
Without being limited to theory, it is believed that the prior art does not describe that herein relevant type III PKS octaketide synthases (OKS) may be active in vivo in a heterologous production host cell of a different genus, e.g. a plant type III OKS may be able to create non-reduced octaketides in vivo in a heterologous production host cell, such as e.g. a recombinant Aspergillus production host cell.
The patent application PCT/EP2014/078540 was filed 18 Dec. 2014. At the filing/priority date of the present patent application PCT/EP2014/078540 was not published. It describes a glycosyltransferase (GT) isolated from Dactylopius coccus costa insect which is capable of: (I): conjugating glucose to flavokermesic acid (FK); and/or (II): conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make carminic acid.
PCT/EP2014/078540 does not directly and unambiguously describe herein discussed relevant non-reduced octaketides and/or polyketide synthases (PKS).